Macrophages are one of the principal cells of the immune system and play a major role in innate immunity. They express pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs). Pattern recognition receptors allow the macrophage to bind, internalize (phagocytosis), and destroy pathogens displaying PAMPs. Chronic inflammation resulting from a maladaptive response of the immune system to a variety of insults or challenges is now recognized to play a role in many chronic diseases, e.g., atherosclerosis and type 2 diabetes. The macrophage plays a central role in these diseases. In atherosclerosis, macrophages enter the arterial intima where they take up oxidized LDL particles and efflux the internalized cholesterol to apoA1 and HDL particles for transport to the liver and excretion. Macrophages use a subset of pattern recognition receptors termed scavenger receptors to bind oxidized LDL particles for phagocytosis. In type 2 diabetes numerous classically activated macrophages (M1 polarization) take residence in adipose tissue where they secrete pro-inflammatory cytokines resulting in decreased insulin sensitivity and predispose to development of type 2 diabetes. We recently discovered that carboxylesterase 1 (CES1), a candidate enzyme for hydrolyzing stored cholesteryl esters to generate free cholesterol for efflux from the macrophage, affects macrophage gene expression. Specifically, silencing CES1 in THP1 macrophages resulted in a dramatic decrease in CYP27A1 expression, a cytochrome P450 enzyme that oxidizes cholesterol to generate 27-OH cholesterol (27-OHCh), and in a decrease in LXR? expression. In addition to CYP27A1 and LXR?, in lipid loaded THP1 macrophages with CES silenced, the expression of scavenger receptor (SR)-A, CD36, and ABCA1 is reduced. The expression of CYP27A1 is controlled by the nuclear receptor heterodimers PPAR?/RXR and RAR/RXR. Both of these heterodimers are permissive thus they can be activated by ligands of either of the protein components. PPAR?/RXR also controls CD36 and LXR? expression. The product of CYP27A1, i.e. 27-OHCh, is a ligand for the nuclear receptor LXR? a component of the heterodimer LXR?/RXR that regulates expression of the cholesterol transporters responsible for efflux to apoA1 and HDL, namely ABCA1 and ABCG1, respectively. These results suggest that CES1 silencing alters signaling through PPAR?/RXR and/or RAR/RXR as well as LXR?/RXR. Therefore, our hypothesis is that CES1 participates in the regulation of CYP27A1 in macrophages by affecting the availability of ligands needed by PPAR?, RXR?, and/or RAR?. We plan to investigate this hypothesis with three specific aims: Aim 1. Characterize the role of nuclear receptors in the alterations in macrophage gene expression caused by either CES1 knockdown or CES1 overexpression; Aim 2. Discover endogenous pro-ligands that are metabolized by CES1, which liberates ligands for nuclear receptors; and Aim 3. Determine whether silencing CES1 in THP1 macrophages affects activation state (M1 vs. M2 polarization), cytokine production, and phagocytosis. The work proposed here is innovative because it will delineate the novel molecular mechanisms by which CES1 regulates CYP27A1 transcription and the broader network of lipid metabolism and inflammatory genes. Beyond transcriptional and phenotypic alterations in macrophages, the proposed studies will also identify natural ligands for PPAR? and RAR using innovative and complementary metabolomic strategies. The work is significant because it will yield a fundamental understanding of the role of CES1 in shaping macrophage phenotype and function, including the regulation of phagocytosis and cytokine production during macrophage activation.